Aminoglycoside (AGs) antibiotics are used worldwide because of their potent antimicrobial activities and low cost. They are widely used despite the significant side effects of ototoxicity and nephrotoxicity. Recent evidence from independent laboratories demonstrated that AGs accumulate rapidly in hair cells because of their ability to enter these cells through the mechanoelectric transducer channel located near the tops of the stereocilia. Channel biophysical properties promote entry through the channel but limit exit through these same channels. Our data clearly show that ototoxicity can be prevented by blocking entry via these channels. The goal of this proposal is to develop novel non-ototoxic aminoglycosides. Our team has unique insights into the biophysical properties of the mechanically gated channels and can use this knowledge to design compounds that are sterically and/or electrically restricted from entering the channel and therefore the hair cell. Sites for modification are selected on the AG backbone so as not to interfere with antimicrobial activity. We have in hand the ability to investigate these compounds at the channel, cellular, end organ, system and whole animal level, using electrophysiological, optical, molecular and pharmacological means to monitor ototoxicity as well as antimicrobial activity. Complementary to the development of these compounds will be identifying the mechanism of entry of the AGs into the endolymph compartment. Upon identification of this pathway we will devise means to limit transport of existing AGs so that a co-treatment plan might prevent access of the AGs to the MET channel and thus limit entry into hair cells. The focus of this proposal is to design treatment regimes, either by creating novel AGs, or co-treatment plans, that will ameliorate ototoxicity due to AG administration. At the end of the proposed two-year research period, we will have applied our unique knowledge base and skill sets to gain insights into the effectiveness of either of these approaches in reducing ototoxicity caused AGs. PUBLIC HEALTH RELEVANCE: Aminoglycosides (AGs) are the most widely used antibiotics worldwide, despite having a high incidence of ototoxicity. We have shown that auditory hair cells are sensitive to AGs because of their high rate of AG uptake, arising from the ability of AGs to pass through a novel mechanosensitive ion channel located at the tops of the stereocilia. Our plan is to design new antibiotics that are sterically and/or electrically restricted from passing through this channel. In conjunction with this approach we will identify the mechanism by which AGs enter the endolymph solution and devise a cotreatment plan to block the responsible transport mechanism, thus preventing the AGs from reaching the ion channel that they permeate.